DESCRIPTION: This second revision of an R01 application has been substantially rewritten and is designed to study the leukemogenic role of a fusion transcription factor termed CBF beta-MYH11. The Investigator proposes three Specific Aims. In the first Specific Aim, he will follow up on his preliminary studies in which expression of the CBF beta-MYH11 gene in 32D cl3 cells led to apoptosis. Several cell lines already developed in the Applicant's lab undergo apoptosis upon shifting from IL-3 to G-CSF, distinctly different from that seen in the parental 32D cl3 cells. The mechanism by which apoptosis is induced will be studied by generating additional cell lines. The effects of the CBF beta-MYH11 gene on leukemogenesis will be studied by coexpressing a number of genes known to suppress apoptosis. The Investigator plans to express a p53 mutant, or BCL2 with the hope of suppressing the apoptosis seen in the CBF beta-MYH11 expressing cells. In a recently submitted update letter, the Investigator has now shown that the G-CSF receptor is down-modulated in these cell lines, suggesting an alternative course of investigation, namely coexpressing the G-CSF receptor using a powerful promoter. Furthermore, the update letter also suggested that raf-1 expression may prevent apoptosis. Using these methods, the Investigator hopes to develop a cellular model by which CBF beta-MYH11 induces a transformed phenotype. In the second specific aim, the mechanism by which this fusion gene operates will be explored. Two alternative hypotheses are presented for testing. The first is that CBF beta-MYH11 is oncogenic because it down-modulates a number of critical regulatory genes; alternatively it is oncogenic by activating critical genes. By expressing a dominant negative mutant of the normal CBF beta partner, PEBP2 alpha, the Investigator hopes to determine whether either down-modulation or up-modulation of target genes is critical for the transformed phenotype. Once repression or enhancement is identified, additional experiments are designed to further understand this mechanism. A subtractive library strategy will be employed to identify the genomic targets of the fusion gene. This will be accomplished by expressing an inducible fusion gene, based on the estrogen receptor, the metallothionine promoter, or the MMLV promoter. Genes obtained by the differential screen will first be used to confirm their appropriate regulation in response to the fusion gene expression, and then will be further explored. The third Specific Aim of the is to identify domains of the fusion gene required to alter the growth properties of target cells. Site directed mutagenesis will be employed in an attempt to identify either a trans activating domain of the fusion gene, or a region which alters the binding properties of the normal transcription factor partners. Cellular localization of the fusion gene will also be explored in this Specific Aim.